The ultimate goal of the Human Genome Project was to determine the sequence of the 3 billion "genetic elements", or nucleotides, that make up the human genome. The human sequence of the human genome is of enormous value and promises to revolutionize biological research and clinical medicine. It also comes with the realization that the complete genome sequence is only a beginning in our understanding of human biology. One of the greatest mysteries is the regulation of genes present within each cell. Despite being genetically identical, cells from different tissues look differently and perform very different functions. For example, although dopamine system genes are present in the cells of brain, muscle, and skin, such genes are active in neurons but not in muscle or skin cells. It is now believed that tissue-specific expression is achieved by the epigenetic regulation of genes via processes such as DNA methylation. More specifically, one of the nucleotides, namely cytosine, can be present in two functional states methylated or unmethylated. Methylated cytosines are sometimes referred as the 5th base of human DNA. DNA methylation profiles are highly variable across different cells, even in the same organism, and such variation depends on tissue, age, sex, diet, and numerous other factors. Over the last decade a series of new methods have been developed to investigate DNA methylation profiles across large DNA regions - chromosomes and even entire genomes. Unfortunately, all these methods exhibit significant limitations as they require large amounts of DNA, interrogate only a small fraction of methylatable nucleotides or are able to scan only short DNA fragments. This project is dedicated to development of a new technology for genome wide DNA methylation, or methylome, analysis. The key innovation consists of a combination of two powerful technologies: targeted deposition of extended groups of biopolymers on DNA, and the application of microarrays. Engineered DNA methyltrasferases, enzymes that methylate DNA, will be used to attach fluorescent labels on unmethylated cytosines, and these labeled DNA fragments will be interrogated on tiling microarrays. Microarrays are small pieces of glass containing millions of short DNA sequences that will hybridize to and highlight the unmethylated DNA fragments. This new approach exhibits numerous advantages over the existing methods for DNA methylation profiling in terms of simplicity, sensitivity, informativeness, and robustness. This technology may significantly contribute to our understanding of development, tissue differentiation, aging, and the molecular basis of complex disease, among numerous other fundamental questions of the life sciences.